INTRODUCTION
Microvascular rarefaction is critical in the development of kidney fibrosis.(Wu CF, Duffield JS, Lin SL Am J Pathol. 2013 [1] (full text)). Pericytes regulate medullary and cortical blood flow; after injury they become an important source of tubule-interstitial myofibroblasts.(Humphreys BD, Bonventre JV. Am J Pathol. 2010 [2]). However, their involvement in the early phase of I/R injury has been poorly investigated.
MATERIAL AND METHODS
Ten pigs underwent to 30 min of renal warm Ischemia, followed by 24h of Reperfusion. Five pigs were treated with C1-Inhibitor (C1-Inh). Biopsies were analyzed by IHC for PDGFRβ and Caspase3. AnnV/IP analysis, FACS and IF were performed on human placental Pericytes stimulated with C5a (1×10-7M) and TGF-β1 (10ng/ml, as positive control for Pericytes Transdifferentation) for 24h.
RESULTS
I/R injury led to pericyte phenotype dysfunction as indicated by a significant reduction of PDGFRβ expression in peritubular capillaries (Figura 1). Immunohistochemical analysis showed the loss of Pericytes at T60 (B) and T24 (C) respect to T0 (A). Treatment with C1-Inh for 24h restored PDGFRβ expression (F) compared to untreated (C), (*p<0.05). When activated, capillary and perivascular PDGFRβ+-pericytes upregulated a typical fibroblast marker α-SMA (red) (Figura 2) (B). C1-Inh treatment preserved the physiological phenotype (C). In accordance, in vitro C5a significantly induced pericytes- myofibroblasts trans-differentiation (Figura 3) (A), with significant down-regulation of PDGFRβ (D)(* p<0.05), evident remodeling of α-SMA in stress fibers and increased Collagen I expression (E), indicating the acquirement of a contractile myofibroblasts phenotype. Interestingly, both in vivo than in vitro, I/R and Complement did not induce pericytes apoptosis. (Figura 4 left) Serial renal section showed PDGFR β+ and Caspase3– cells at early time after reperfusion. In according with this finding, AnnexinV/PI analysis did not reveal C5a induced-apoptosis and necrosis (Figura 4 right). In conclusion, I/R was associated with a significant decrease in capillary lumen area (Figura 5).(B), which result in an overall microvascular rarefaction (A). Complement Inhibition restored basal capillary area fraction.
CONCLUSIONS
During renal I/R injury, Complement caused Pericytes-Myofibroblasts transdifferentation, leading to a vasoconstriction of microvessels and fibrosis. C1-Inh may be an effective therapeutic strategy to prevent Pericytes loss in transplanted kidney.